Glycolipid flip-flop across the endoplasmic reticulum during protein N-glycosylation.

摘要

The majority of proteins synthesized in the endoplasmic reticulum (ER) are glycoproteins. From a process involved in cell wall synthesis in archaea and some bacteria, N-glycosylation has evolved into the most common covalent protein modification in eukaryotic cells. The sugars are added to nascent proteins as a core oligosaccharide unit, which is then extensively modified by removal and addition of sugar residues in the ER and the Golgi complex. It has become evident that the modifications that take place in the ER reflect a spectrum of functions related to glycoprotein folding, quality control, sorting, degradation, and secretion. The glycans not only promote folding directly by stabilizing polypeptide structures but also indirectly by serving as recognition "tags" that allow glycoproteins to interact with a variety of lectins, glycosidases, and glycosyltranferases.;The biosynthesis of glycoproteins is a task shared by the ER and the Golgi apparatus. Assembly of the core glycan is the product of a biosynthetic pathway in which monosaccharides are added to a lipid carrier (dolichol pyrophosphate) by glycosyltransferases in the ER membrane. Seven sugars are added on the cytoplasmic surface after which the sugar moiety is translocated (flipped) to the lumenal side. Mannosyl phosphoryl dolichol and glucosyl phosphoryl dolichol are the sugar donors in the ER lumen that are synthesized on the cytoplasmic leaflet of the ER. Flipping of all such essential glycolipids is catalyzed by ATP-independent, bi-directional ER-resident flippases. The chapters in this thesis document our efforts to develop biochemical assays for measuring glycolipid flipping in vitro with the broader aim of identifying this class of proteins and elucidate the mechanism of lipid translocation.